beta-Glucocerebrosidase activity in mammalian stratum corneum

Although previous studies have demonstrated a crucial role for the enzyme b eta-glucocerebrosidase (GlcCer'ase) iu the final steps of membrane structur al maturation in mammalian stratum cornuem (SC) and epidermal homeostasis, the precise in vivo localization of GlcCer'ase activity and protein is not known. Here, we developed a fluorogenic in situ assay on histologic section s (zymography) to elucidate the in vivo distribution of GlcCer'ase activity , and further characterized and localized the SC GlcCer'ase activity in vit ro, The zymographic technique revealed higher GlcCer'ase activity in upper stratum granulosum and SC, both in murine and human SC; activity that was b oth inhibited by conduritol B epoxide, a specific GlcCer'ase inhibitor, and pH-dependent; i.e., present at pH 5.2, and absent or significantly reduced at neutral pH (7.4), consistent with the known pH optimum for epidermal Gl cCer'ase in vitro. Immunohistochemical staining for GlcCer'ase protein show ed enhanced fluorescent signal in the outer layers of human epidermis, conc entrated at the apex and margins of stratum granulosum and lower SC, Moreov er, in extracts from individual epidermal layers, GlcCer'ase activity was p resent throughout murine epidermis, with the highest activity in the SC, pe aking in the lower-to-mid-SC. The SC activity was stimulated >10-fold by so dium taurocholate, and inhibited by bromoconduritol B epoxide, Finally, iso lated membrane couplets, prepared from SC sheets, also demonstrated signifi cant GlcCer'ase activity. These data localize GlcCer'ase activity to the ou ter epidermis by three different techniques, and support the role of this e nzyme in extracellular processing of glucosylceramides to ceramides, requir ed for permeability barrier maturation and function.